N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride) (W-7) causes increases in intracellular free Ca²⁺ levels in bladder female transitional carcinoma (BFTC) cells

The effect of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride) (W-7), a widely used calmodulin inhibitor, on intracellular free Ca²⁺ levels ([Ca²²+]_i) in bladder female transitional cancer (BFTC) cells was examined using fura-2 as a Ca²⁺ dye. W-7 (10-1000 μM) caused an increase in [Ca²⁺]_i in a concentration-dependent manner with an EC50 of 75 μM. The [Ca²⁺]_i response was composed of an initial rise and a sustained plateau without significant decaying during the measurement of 250 seconds. Extracellular Ca²⁺ removal dramatically decreased the Ca²⁺ signals by 50-90%. W-7 (100 μM) failed to induce a [Ca²⁺]_i increase in Ca²⁺-free medium after pretreatment with thapsigargin (1 μM), an endoplasmic reticulum Ca²⁺ pump inhibitor; conversely, W-7 pretreatment abolished the Ca²⁺ release induced by thapsigargin. Addition of 3 mM Ca²⁺ increased [Ca²⁺]_i after preincubation with 100 μM W-7 in Ca²⁺-free medium. W-7-induced Ca²⁺ release was not altered by inhibiting phospholipase C with 2 μM 1-(6-((17b-3-methoxyestra-1,3,5 (10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) (U73122). Together, this study shows that W-7 caused [Ca²⁺]_i increases in human bladder cancer cells by releasing intracellular Ca²⁺ from the endoplasmic reticulum and also by causing extracellular Ca²⁺ influx with the later playing a dominant role. The W-7 induced intracellular Ca²⁺ release was uncoupled to a prior elevation in intracellular levles of inositol 1,4,5-trisphosphate (IP₃).